This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our use of the mass spectrometry core facility will be to support our efforts in proteinase inhibitor discovery, characterization, and design. Our lab studies three major classes of proteinases as potential therapeutic targets: serine (membrane type serine protienase 1, kaposi's sarcoma herpes virus proteinase, and the granzymes), cysteine (cruzain, SARS major proteinase, falcipain), and aspartyl proteases (eqolysin, and HIV proteinase). Our work involves developing robust expression systems, enzymatic and biochemical characterization, determining peptide substrate specificity, synthesizing effective fluorogenic substrates, developing high-throughput screening assays, screening candidate inhibitor libraries, synthesizing novel inhibitors, and characterizing the mechanism of action of these inhibitors kinetically and structurally. The use of the mass spectrometry core facility is integral to our efforts. Mass spectrometry provides an orthogonal and definitive way of confirming that a given inhibitor forms a covalent adduct in the expected manner with the proteolytic enzyme under investigation.